T. Kokubo et al./ Biomaterials 24(2003)2161 2163 components for it to have apatite-forming ability and for it to be able to integrate with bone in the body. However, assessments of apatite formation on material with different compositions in SBF imply that CaO and P2O5 are not the essential components for apatite formation. Fig 3 shows the compositional dependence of apatite formation in SBF of glasses in the systems Cao-P2O5-SiO2 [25]. Na20-CaO-SiO2 [26], and K20-SiO -T10, [27]. Interestingly, in the Cao-P2O5- SiO2 system, the composition of the glass forming an apatite layer in an SBF was based on the Cao-sio system and not on the Cao-P2Os system. The P2Os-free Fig. 2. TEM photograph of the interface between glass-ceramic A-w Cao-SiO2 glasses were actually shown to bond to living and a rat tibia(8 weeks after implantation) bone in animals by forming apatite on their surfaces [28]. Evaluation of the Na, O-CaO-Sio, system indi Table 2 cates that, not only the P2Os-free Cao-Sio2 glasses, but lon concentrations of human blood plasma, SBF and m-SBF also the CaO- and P2Os-free Na,O-SiO2 glasses can form apatite in SBF. furthermore even the k0-TiO based glasses have been shown to form apatite. On Na K Mg Ca-CI HCO3 HPOA SO4 glasses such as those in the binary systems, CaO-Sio ood plasma 142.0 5.0 1.5 5103.027.01.00.5 Na2O-SiO2, and K2O-TiO2, apatite formation is SBF 14205.01.52.5148.84.21.0 speculated to proceed by the following mechanism m-SBE 14205.01.52.5103.010.01.00.5 The glass releases Ca, Na*, or K ions from its Buffered at pH 7.40 with tris-hydroxymethylaminomethane and surfaces via an exchange with the h3o ion in the sBF IM HCI to form Si-OH or Ti-OH groups on their surfaces b Buffered at pH 7.40 with 2-(4-(2-hydroxyethyl)-l-piperazinylethane Water molecules in the sbF then or simultaneously sulfonic acid and IM NaOH react with the Si-O-Si or Ti-O-ti bond to form additional Si-OH or Ti-OH groups. The Si-OH and When this process occurs, a chemical bond is formed Ti-oH groups formed induce apatite nucleation, and between the bone mineral and the surface apatite to decrease the interfacial energy between them. It can be nucleation by increasing the ionic activity product (IAP) concluded from these findings that an essential require of apatite in the fluid [26, 27, 29]. Once the apatite nuclei are formed, they can grow spontaneously by consuming ment for an artificial material to bond to living bone is the calcium and phosphate ions in the surrounding fluid the formation of a layer of biologically active bone-like apatite on its surface in the body. because the body fluid is highly supersaturated with The in vivo formation of an apatite layer on the respect to the apatite [18,191 surface of a bioactive ceramic can be reproduced in a protein-free and acellular simulated body fluid(SBF 2. 3. Functional groups for apatite nucleation which is prepared to have an ion concentration nearly equal to that of human blood plasma [13-15, 18-24 The catalytic effect of the Si-OH groups and Ti-OH (Na+142.0,K+5.0,Ca2+2.5,Mg2+1,C1-147.8, groups for the apatite nucleation has been proven by the HCO3 4.2, HPO4 1.0, and SO4 0.5 mM, and a ph of observation that silica and titania gels produced by the 7.25 or 7.40), as given in Table 2. Therefore, the sol-gel method form apatite on their surfaces in SBF bioactivity of an artificial material can be evaluated by and these functional groups are abundant on their examining the formation of apatite on its surface in surfaces [30, 31]. Zirconia [32], niobium oxide [33], and SBF. In this case, what types of materials form an tantalum oxide [34] gels have also been shown to form apatite layer on their surfaces in the living body? apatite on their surfaces in SBF, as shown in Fig 4. This indicates that Zr-OH, Nb-OH, and Ta-OH groups are ffective for apatite nucleation. Other assessments using 2. 2. Mechanism of apatite formation on bioactive self-assembled monolayers (SAM)in SBf have indi ceramIcs cated that COOH and PO,H2 groups are also effective for apatite nucleation [35] Representative bioactive ceramics, e. g, Bioglass, However, it has been suggested that the efficacy of HA, and grass-ceramic A-W, contain Ha or its apatite nucleation of the above functional groups is such as CaO and P2Os. Therefore, it had determined, not by their composition alone, but ved that a material should have these in a complicated fashion that is dependent on theirWhen this process occurs, a chemical bond is formed between the bone mineral and the surface apatite to decrease the interfacial energy between them. It can be concluded from these findings that an essential require￾ment for an artificial material to bond to living bone is the formation of a layer of biologically active bone-like apatite on its surface in the body. The in vivo formation of an apatite layer on the surface of a bioactive ceramic can be reproduced in a protein-free and acellular simulated body fluid (SBF), which is prepared to have an ion concentration nearly equal to that of human blood plasma [13–15, 18–24] (Na+ 142.0, K+ 5.0, Ca2+ 2.5, Mg2+ 1.5, Cl 147.8, HCO3 4.2, HPO4 2 1.0, and SO4 2 0.5 mm, and a pH of 7.25 or 7.40), as given in Table 2. Therefore, the bioactivity of an artificial material can be evaluated by examining the formation of apatite on its surface in SBF. In this case, what types of materials form an apatite layer on their surfaces in the living body? 2.2. Mechanism of apatite formation on bioactive ceramics Representative bioactive ceramics, e.g., Bioglasss, HA, and grass-ceramic A-W, contain HA or its components, such as CaO and P2O5. Therefore, it had been believed that a material should have these components for it to have apatite-forming ability and for it to be able to integrate with bone in the body. However, assessments of apatite formation on materials with different compositions in SBF imply that CaO and P2O5 are not the essential components for apatite formation. Fig. 3 shows the compositional dependence of apatite formation in SBF of glasses in the systems: CaO–P2O5–SiO2 [25], Na2O–CaO–SiO2 [26], and K2O–SiO2–TiO2 [27]. Interestingly, in the CaO–P2O5– SiO2 system, the composition of the glass forming an apatite layer in an SBF was based on the CaO–SiO2 system and not on the CaO–P2O5 system. The P2O5-free CaO–SiO2 glasses were actually shown to bond to living bone in animals by forming apatite on their surfaces [28]. Evaluation of the Na2O–CaO–SiO2 system indi￾cates that, not only the P2O5-free CaO–SiO2 glasses, but also the CaO- and P2O5-free Na2O–SiO2 glasses can form apatite in SBF. Furthermore, even the K2O–TiO2- based glasses have been shown to form apatite. On glasses such as those in the binary systems, CaO–SiO2, Na2O–SiO2, and K2O–TiO2, apatite formation is speculated to proceed by the following mechanism. The glass releases Ca2+, Na+, or K+ ions from its surfaces via an exchange with the H3O+ ion in the SBF to form Si–OH or Ti–OH groups on their surfaces. Water molecules in the SBF then or simultaneously react with the Si–O–Si or Ti–O–Ti bond to form additional Si–OH or Ti–OH groups. The Si–OH and Ti–OH groups formed induce apatite nucleation, and the released Ca2+, Na+, or K+ ions accelerate apatite nucleation by increasing the ionic activity product (IAP) of apatite in the fluid [26,27,29]. Once the apatite nuclei are formed, they can grow spontaneously by consuming the calcium and phosphate ions in the surrounding fluid because the body fluid is highly supersaturated with respect to the apatite [18,19]. 2.3. Functional groups for apatite nucleation The catalytic effect of the Si–OH groups and Ti–OH groups for the apatite nucleation has been proven by the observation that silica and titania gels produced by the sol–gel method form apatite on their surfaces in SBF, and these functional groups are abundant on their surfaces [30,31]. Zirconia [32], niobium oxide [33], and tantalum oxide [34] gels have also been shown to form apatite on their surfaces in SBF, as shown in Fig. 4. This indicates that Zr–OH, Nb–OH, and Ta–OH groups are effective for apatite nucleation. Other assessments using self-assembled monolayers (SAM) in SBF have indi￾cated that COOH and PO4H2 groups are also effective for apatite nucleation [35]. However, it has been suggested that the efficacy of apatite nucleation of the above functional groups is determined, not by their composition alone, but in a complicated fashion that is dependent on their Table 2 Ion concentrations of human blood plasma, SBF and m-SBF Concentration/mm Na+ K+ Mg2+ Ca2+ Cl HCO3 HPO4 2 SO4 2 Blood plasma 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5 SBFa 142.0 5.0 1.5 2.5 148.8 4.2 1.0 0.5 m-SBFb 142.0 5.0 1.5 2.5 103.0 10.0 1.0 0.5 aBuffered at pH 7.40 with tris-hydroxymethylaminomethane and 1m HCl. bBuffered at pH 7.40 with 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethane sulfonic acid and 1m NaOH. Fig. 2. TEM photograph of the interface between glass-ceramic A-W and a rat tibia (8 weeks after implantation). T. Kokubo et al. / Biomaterials 24 (2003) 2161–2175 2163